Anionic polysaccharides and oligosaccharides occur naturally in the form of glycosaminoglycans, such as heparin, dermatan sulfate, chondroitin sulfate, and the like. In addition, synthetic or natural carbohydrates can be converted to charge-bearing species by derivatization, especially to the multiplicity of free hydroxyl groups by, for example, sulfation. Oligosaccharides, as they occur natively, or fragments of these oligosaccharides, are notoriously difficult to separate on a preparative scale. Satisfactory resolution is sometimes achieved using such techniques as polyacrylamide gel electrophoresis or capillary zone electrophoresis, but, generally, only nanogram or microgram amounts can be obtained conveniently. This is inherent in the nature of capillary zone electrophoresis (Fujiwara, S., et al., Anal Chem (1987) 59:487-490; Al-Hakim, A., et al., Anal Biochem (1991) 195:68-73; Carney, S. L., et al., Ibid. 132-140). In the case of polyacrylamide gel electrophoresis, this low capability for recovery results from the necessity to use high concentrations of about 30-40% polyacrylamide in the gel (Rice, K. D., et al., Biochem J (1987) 244:515-522; Knudson, W., et al., Biochemistry (1984) 23:368-375). Another technique, reversed-phase ion-pairing HPLC, is inconvenient on a preparative scale because of the large amounts of organic solvents and ion-pairing reagent required to elute the oligosaccharides (Guo, Y., et al., Analyt Biochem (1989) 168:54-62; Guo, Y., et al., Anal Biochem (1989) 175:96-104).
Anion exchange has also been used for oligosaccharide separation in the context of HPLC using "strong" anion-exchange supports containing quarternary amine functional groups (Bienkowski, M. J., et al., J Biochem (1985) 260:256-265; Merchant, Z. M., et al., Biochem J (985) 229:369-378). The problem with strong anion-exchange supports resides in poor resolution and the need for high salt concentrations to elute the adsorbed anionic oligosaccharides. However, cellulose supported anionic exchange supports have been used to remove heparin from blood plasma (EP application 281,128).
It has now been found, surprisingly, that supports coated with polyethyleneimine (PEI) provide excellent resolution of anionic carbohydrates on a preparative scale. While PEI-coated supports have been used for separation of peptides and proteins and for oligonucleotide separations, the only use of any similar technique with respect to anionic oligosaccharides has been with respect to thin-layer chromatography, i.e., for analytical, not preparative, purposes (Ram, P. A. et al., Anal Biochem (1989) 178:421-426; Zagrod, M. E. et al., Invest Opthamol Vis Sci (1985) 26:1475-1473). PEI has also been used as a detection for histological staining to determine the presence of anionic substances, including heparin in biological samples. See, for example, Whiteside, C. et al., Lab Invest (1989) 61:650-660; Spooner, B. S. et al., Arch Oral Biol (1989) 34:541-549. PEI, including PEI cross-linked with diisocyanate has been used to precipitate heparin or other polysaccharides from solution. See, e.g., Belgian patent 888463. To applicants, knowledge, PEI-coated chromatographic supports have not previously been used for either analytic or preparative separation of oligosaccharides.